Post-Transcriptional Modification

• Covalent post-transcriptional modification by conjunction of “prosthetic groups” can help to augment or inhibit protein function
  • Examples of post-transcriptional modification:
    • Phosphoserine, phosphotyrosine, phosphothreonine are the most frequent modified amino acids in protein and help modulate cellular functions.
      • Requires phosphokinase enzymes
    • Hydroxyl group addition to proline and lysine stabilizes collagen fibers
      • Lack of vitamin C (ascorbate) results in scurvy as Vitamin C helps collagen hydroxylation process of proline and lysine
        • Vitamin C is an essential cofactor to prolyl hydroxylase enzyme
    • Acetyl group to the amino terminal helps resist degradation
    • Ubiquitin tags proteins for degradation.
    • Glychosylation, methylation, sumoyolation, ubiquitination, etc. modifications are relatively new and roles are unclear

Collagen

• More than 30% of protein in the human body is some form of collagen structural protein
• Collagen is composed of 33% glycine and 20% proline or hydroxyproline
  • Also contain amounts of lysine, hydroxylysine, and tyrosine.
  • No tryptophan or cystiene in collagen
• α-collagen – basic polypeptide procollagen unit with left-hand helical structure
  • Most helices are right-handed
• β-collagen – double helix of procollagen
• γ-collagen – triple α-helix of procollagen, formed in the ER
• Collagen fibrils are arranged differently in various tissues:
  • Tendon – in parallel bundles
  • Skin – in sheets of fibrils layered at many angles
  • Cartilage – no distinct arrangement
  • Cornea – planar sheets stacked crossways to minimize light scatter

Stability of Proteins

• Proteins are only marginally stable because the free energy difference between a typical folded and unfolded protein is very small
• Marginal stability is biologically advantageous, providing flexibility for protein function
  • Enzymes undergoing conformational changes to induce fit of a specific substrate
• 30%-40% of all proteins in the body can perform different tasks, binding to more than one molecule depending on different task requirements

Studying Protein Function

• To understand protein function, investigators must:
  • Purify the protein of interest
  • Determine the primary amino acid sequence

Purification of Protein

• Purification is necessary to determine the amino acid sequence of a specific protein
  • Determine family of proteins – protein function can be inferred from this information
  • Can compare protein between different organisms to determine evolutionary relationship
    • Similar proteins are likely to be essential to life
  • Knowledge of amino acid sequence can help determine biochemical function
    • Some amino acid sequences have been identified as signals for destination or processing of that protein, i.e. membrane or nuclear localization
    • Can develop antibodies against protein to further improve purification or allow clinical investigation
    • Amino acid sequence can be used to make DNA probes for the genetic sequence for study
• Purification of protein can allow crystallography and x-ray data to help determine the tertiary form

Purification Steps

• Differential centrifugation of a homogenate of a cell to isolate different fractions of the cell
  • Isolate nuclear faction, mitochondrial faction, or microsomal faction
• Purification can be based on solubility, size, charge, or binding affinity
  • Dialysis can separate larger molecules from smaller molecules
    • Exploits a semi-permeable membrane and allowing the concentrated solution to equilibrate with the buffer
  • Gel filtration can be used to discriminate by size
    • Larger molecules take longer to pass through beads, so different fractions can be obtained and tested for activity
  • Chromatography:
    • Affinity chromatography can separate proteins based on a specific binding affinity for a molecule using a column matrix
      • Preferred choice if antibody is available because it can provide very specific enrichment
    • Ion exchange chromatography separates proteins based on net charge at a specific pH
    • Salting out changes the salinity at which a protein becomes insoluble and precipitates out
    • High pressure liquid chromatography (HPLC) – can be used for almost all column chromatography by applying pressure to achieve higher resolving power and rapid separation.
• Check for effectiveness of purification process:
  • Check yield or total protein content in assay solution
    • Gel electrophoresis separates proteins by charge, shape, or molecular mass
      • SDS-PAGE – polyacrylamide gel electrophoresis
        • Contains a large negative charge and sticks to proteins
        • Allows proteins to have the same charge but different molecular weight for separation
  • Check specific activity of protein
  • Example: LDH (lactate dehydrogenase) changes lactate and NAD+ to pyruvate, NADH and H+
    • Measure the yield of its product to determine activity, in this case, NADH is easiest to measure
• Other purification techniques:
  • Isoelectric focusing electrophoresis
    • Separation by pI (isoelectric point) of the protein, when they have no net electric charge
    • Isoelectric focusing gels are formed contained a pH gradient which the protein solution is subjected to electrophoresis without SDS
  • Two-dimensional electrophoresis
    • Combine isoelectric focusing with SDS-PAGE to obtain very high resolution separation of proteins.
    • After SDS-PAGE separation, purified protein can be cut out and subjected to mass spectrometry
  • Mass Spectrometry – determine idenity of protein by mass fragment pattern

Determination of Amino Acid Sequence

• Determine look at amino acid composition
  • Hydrolyze protein to break up protein to amino acids at 110°C in 6M HCL for 24 hours
  • Separate by ion-exchange chromatography
  • Use ninhydrin to measure absorbance
• Edman degradation
  • Amino-terminal residue is labeled with phenyl isothiocyanate and cleaved from the protein to be identified by chromatography procedures.
  • Process repeated until complete sequence is revealed.

Proteomes

• Goal of many researchers now is to identify and define proteomes
  • Proteins expressed by the genome that work together in a functional unit
  • Defining proteome requires information encompassing each protein, function of proteins in proteome, and interactions of the protein as a functional unit
    • Can include information from alternatively spliced RNA, post-transcriptional modification, etc. depending on cellular environment at any given moment

Objectives

Objectives - Introduction to Proteins III.doc

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